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Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

Gorska A, Swiatkowska A, Dutkiewicz M, Ciesiolka J - PLoS ONE (2013)

Bottom Line: Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells.A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins.One of these oligomers might be used in the future as a support treatment in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

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RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA.The 5′-end-[32P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′-OMe or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H2O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.
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pone-0078863-g004: RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA.The 5′-end-[32P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′-OMe or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H2O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.

Mentions: A radioactively labelled ΔNp53utr-Luc transcript was incubated with each unmodified or modified antisense oligonucleotide in RRL. In the presence of unmodified oligomer no. 7b and its GAP variant characteristic RNase H cleavages occurred at the predicted sites, in the region G217-G226 (Fig. 4). The 2′-OMe variant did not activate RNase H, as it was expected. In the case of unmodified oligomer no. 1, cleavages appeared in the region between G56 and G60. However, surprisingly, GAP oligomer no. 1 did not trigger RNase H activity. Moreover, all variants of oligomer no. 1 induced cleavages in the region between G161 and G168 which is opposite to their hybridization site in the RNA secondary structure (Fig. 4). To explain this observation, an RNase H assay was performed in the presence of unmodified oligomer no. 1 and its GAP variant using a commercially available enzyme from E. coli (Fig. S2). Both oligomers supported RNase H activity and the predicted cleavage patterns were obtained. However, there were no additional cleavages in the region G161-G168, which were observed in RRL (Fig. 2). This discrepancy might result from a slightly different activity of eukaryotic RNase H which was present in RRL and E. coli RNase H which was used in the test assay [40]. According to an alternative explanation, it might be suspected that binding of each variant of oligomer no.1 to the C50-A68 region resulted in partial unwinding of the double-stranded region at the bottom of hairpin G56-C169. The RNA strand located opposite to the oligomer hybridization site became more flexible. In consequence, it was susceptible to spontaneous breakage or cleavage by some other RNases which might be remaining in RRL.


Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

Gorska A, Swiatkowska A, Dutkiewicz M, Ciesiolka J - PLoS ONE (2013)

RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA.The 5′-end-[32P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′-OMe or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H2O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824000&req=5

pone-0078863-g004: RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA.The 5′-end-[32P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′-OMe or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H2O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.
Mentions: A radioactively labelled ΔNp53utr-Luc transcript was incubated with each unmodified or modified antisense oligonucleotide in RRL. In the presence of unmodified oligomer no. 7b and its GAP variant characteristic RNase H cleavages occurred at the predicted sites, in the region G217-G226 (Fig. 4). The 2′-OMe variant did not activate RNase H, as it was expected. In the case of unmodified oligomer no. 1, cleavages appeared in the region between G56 and G60. However, surprisingly, GAP oligomer no. 1 did not trigger RNase H activity. Moreover, all variants of oligomer no. 1 induced cleavages in the region between G161 and G168 which is opposite to their hybridization site in the RNA secondary structure (Fig. 4). To explain this observation, an RNase H assay was performed in the presence of unmodified oligomer no. 1 and its GAP variant using a commercially available enzyme from E. coli (Fig. S2). Both oligomers supported RNase H activity and the predicted cleavage patterns were obtained. However, there were no additional cleavages in the region G161-G168, which were observed in RRL (Fig. 2). This discrepancy might result from a slightly different activity of eukaryotic RNase H which was present in RRL and E. coli RNase H which was used in the test assay [40]. According to an alternative explanation, it might be suspected that binding of each variant of oligomer no.1 to the C50-A68 region resulted in partial unwinding of the double-stranded region at the bottom of hairpin G56-C169. The RNA strand located opposite to the oligomer hybridization site became more flexible. In consequence, it was susceptible to spontaneous breakage or cleavage by some other RNases which might be remaining in RRL.

Bottom Line: Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells.A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins.One of these oligomers might be used in the future as a support treatment in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

Show MeSH
Related in: MedlinePlus